Infants who are breastfed by their HIV-infected mothers are at risk of acquiring HIV infection through breastfeeding. In the absence of interventions, about 14% of breastfed infants will acquire infection via this route. This relatively low transmission rate is surprising since infants ingest virus-containing breast milk many times a day over 18 or more months. The inefficiency of oral transmission in infants is intriguing. We hypothesize that immunomodulatory components of human breast milk help explain this low rate of transmission. Innate immune factors that influence epithelial integrity may affect transmission by regulating the amount and infectivity of virus in breast milk and/or through passive immune processes in the infant gut preventing the establishment of infection. These factors may also reduce activation leading to slower disease progression in HIV-infected breast-fed infants. We hypothesize that human milk oligosaccharides (HMO), soluble toll-like receptors (sTLR) and other innate immune factors may play a role in preventing oral transmission of HIV via breastfeeding and modulate disease progression. The breastfed infant provides a unique human challenge model to investigate these novel factors. Using existing clinical samples from a cohort of over 900 breastfed infants born to HIV-infected mothers in Zambia, we will characterize HMO, sTLR and other components of mucosal immunity in breast milk and investigate their role in oral HIV transmission in infants. In addition to the already collected clinical data, we will incorporate detailed virologic studies of HIV RNA and DNA shedding in breast milk. We have put together a multi-disciplinary scientific team with specific expertise in innate immunity in mucosal fluids who, combined with the large clinical database and virologic studies, will allow rigorous and powerful epidemiologic analyses to investigate: (1) whether higher concentrations of innate immune factors in breast milk are associated with lower rates of breast feeding- associated HIV transmission, independent of HIV-1 RNA and DNA concentrations in breast milk and especially among infants who have disruptions in the oral mucosa due to teeth development and/or oral thrush; (2) whether innate immune factors in breast milk reduce mucosal viral shedding in breast milk relative to expected shedding based on HIV RNA in peripheral blood and other markers of the severity of maternal disease including CD4 counts; and (3) whether higher concentrations of innate immune factors in breast milk are associated with slower disease progression in HIV-infected children while they are being breastfed but not after they are weaned. These studies will allow an in vivo evaluation of the potential role of anti-HIV innate mucosal immunity that will be informative for potential HIV vaccines that harness mucosal innate immunity.